Microbiology & Immunology - Theses
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ItemHarnessing unconventional T-cells for vaccines and immunotherapies in pre-clinical animal models( 2023-03)Unconventional T-cells represent a heterogenous population of CD3+ T-cells that recognise protein and non-protein antigens through MHC-unrestricted mechanisms. Unconventional T-cells also undergo cytokine- or surface receptor-mediated activation independent of T-cell receptor ligation, a characteristic that is commonly associated with innate immune cells and permits rapid responses to stimuli. These cells have diverse effector responses following activation, including direct cytolytic activity against target cells, proinflammatory cytokine production to mediate other immune responses, and antigen presentation to conventional CD4+ and CD8+ T-cells. Several unconventional T-cell subsets have also been explored as immunotherapeutics, in part due to our ability to readily expand them pharmacologically, with some expressing highly conserved public T-cell receptors. Current knowledge gaps with unconventional T-cell immunotherapies includes our understanding of the frequency and phenotype of therapeutic cells in different tissue compartments, and how this is impacted by changes in pharmacological expansion protocols. Additionally, several unconventional T-cell subsets can augment conventional T- and B-cell responses associated with humoral immunity. However, the contribution of these unconventional T-cell populations to conventional adaptive immune responses against protein vaccines or viral infection is not well understood. The overarching aim of this thesis is to characterize unconventional T-cell in the context of immunotherapeutics and during vaccine-elicited immune responses, in pre-clinical animal models. Vgamma9Vdelta2 T-cells are a subset of unconventional T-cells that recognises endogenous and exogenous phosphoantigens and have garnered significant interest for immunotherapies to treat cancer and infectious diseases. While studies in pre-clinical animal models have shown promise, the clinical efficacy with Vgamma9Vdelta2 T-cell therapy has been limited. In Chapter 2, we characterised the Vgamma9Vdelta2 T-cell population at steady-state and following in vivo pharmacological expansion in pigtail macaques. We found the tissue distribution of pharmacologically expanded Vgamma9Vdelta2 T-cells changed based on the antigen administration route. Additionally, our pharmacological expansion protocol drove marked CCR6 downregulation and granzyme B upregulation in expanded Vgamma9Vdelta2 T-cells. Our results highlight how changes to pharmacological expansion protocols can alter the phenotype and tissue distribution of the expanded cell population, which is important to consider as this will likely impact therapeutic efficacy. Lymph nodes are a critical site of adaptive immune responses and the generation of antigen specific Tfh and BGC cells following vaccination. However, vaccine draining lymph node identification to study these responses is hindered by anatomical variations in lymphatic drainage between individuals, and lymph nodes being arranged in clusters with only a subset draining the vaccine site. To improve the identification of vaccine draining lymph nodes in preclinical animal models, we developed a vaccine strategy to label draining lymph nodes with tracking dyes (Chapter 3). We show that protein vaccines co-formulated with tattoo ink accurately labels vaccine draining lymph nodes in both mice and nonhuman primates. Ink-containing lymph nodes had higher frequencies of antigen specific BGC and Tfh cells compared to lymph nodes without ink. Furthermore, the ink coformulation was compatible with flow cytometry-based assays and did not alter the vaccine immune response serologically or at the B- and T-cell level. Unconventional T-cells are capable of humoral immune responses through multiple mechanisms including conventional antigen presentation, co-stimulatory signalling to Tfh cells, and providing both cognate and non-cognate B-cell support. Whether different unconventional T-cell subpopulations significantly contribute to the humoral immune response following vaccination or viral infection has not been well established. In Chapter, 4, we evaluated the contribution of unconventional T-cells to conventional adaptive immune responses elicited by vaccines or influenza infection, using transgenic mice that individually lack gamma delta T-cells, MAIT cells, and NKT cells. We found transgenic animals had comparable serological, Tfh, and BGC responses following immunisation with clinically-relevant vaccine formulation or subclinical influenza infection. Our findings indicate these unconventional T-cell subpopulations are not individually essential for mounting a robust humoral immune response to protein vaccines or viral infection. These results also raise questions about compensation between these unconventional T-cell populations, or a potential lack of unconventional T-cell recruitment to vaccine- or viral-mediated immune responses. Collectively, we evaluated unconventional T-cells in the context of immunotherapies and conventional humoral immune responses, in preclinical animal models. Better animal model characterisation will likely improve clinical translatability of candidate vaccines and therapeutics. Future utilisation of and improvements to the labelling techniques described here will help interrogate vaccine responses in regional draining lymph nodes in preclinical animal models. Our findings also raise important questions about modifying in vivo Vgamma9Vdelta2 T-cells treatment protocols to improve therapeutic cell delivery to target tissues, and modifying vaccine formulations to better recruit different unconventional T-cell populations as part of the humoral immune response.